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be used for control measurements and also for fusion to other proteins to measure
clustering. For the reference measurements, several constructs can be used; for cy-
toplasmic proteins, mGFP is fused to FKBP: 1xFKBP-mGFP or 2xFKBP-mGFP.
FKBP encoding cDNA can be obtained from the vector pC
4
-Fv1E (pC
4
-1xFKBP,
Ariad, now the Clontech iDimerize
™
Inducible Homodimer System). FKBP can
be dimerized by binding of the ligand AP20187. As a result, 1xFKBP-mGFP will
dimerize, while a 2xFKBP-mGFP construct will form trimers, tetramers, etc.
(
Fig. 16.1
). For calibration of EGFR clustering, three different constructs were made:
EGFR-mGFP, EFGR-FKBP-mGFP, and EGFR-2xFKBP-mGFP. All these con-
structs were used for transient transfections. For the production of stable cell lines,
the constructs were subcloned into a pcDNA3.1 vector, which contains a Zeocin
resistance gene. All constructs were amplified in
E. coli
.
16.2.2
Cell lines
All EGFR-expressing cells can be used for measuring EGFR clustering using homo-
FRET. We normally use HeLa and A431 cells and NIH3T3 clone 2.2 cells that do not
express EGFR, which were transfected with human EGFR (Her14 cells). All cells
were cultured at 37
C and 5% CO
2
in Dulbecco's modified eagle's medium
(PAA, Cambridge) containing 7% fetal bovine serum albumin, 2 mM
L
-glutamine,
100 U/ml penicillin, and 100
g/ml streptomycin. For the establishment of stable cell
lines expressing FKBP-mGFP, 2xFKBP-mGFP, EGFR-mGFP, EGFR-FKBP-
mGFP, and EGFR-2xFKBP-mGFP, the cells were transfected with 2
m
m
g plasmid to-
gether with 6
l FuGENE-6 according to the manufacture's protocol and then grown
under selective conditions (500
m
m
M Zeocin) for at least 8 weeks.
16.2.3
Microscope
16.2.3.1
Confocal time-resolved and steady-state fluorescence
anisotropy imaging microscopy
A confocal microscope (Nikon C1, Japan) was equipped with a 473 nm solid-state
diode laser (Becker & Hickl, BDL-473-SMC) with a pulse width of 60 ps and a rep-
etition rate of 80 MHz. The laser light was polarized by a linear polarizer (Meadow-
lark, Frederick, CO, USA) and focused by a 60
water immersion objective (N.A.
1.2, Nikon). The high NA objectives, like the one used here, result in reduced values
for the anisotropy. This does not, however, affect the cluster sizes when reference
constructs are used to calibrate cluster sizes. Fluorescence emission was selected
by a 515/30 nm band-pass filter. The emitted light was then split in a parallel and
a perpendicular channel with respect to the excitation light by a broadband polarizing
beam splitter cube (PBS, OptoSigma, Santa Ana, CA, USA). Signals were detected
with two high quantum efficiency photomultiplier tubes (Hamamatsu H7422P-40)
connected to two time-gated fluorescence lifetime imaging modules (LIMO (
De
Grauw & Gerritsen, 2001
), Nikon Instruments BV, Badhoevedorp, the Netherlands).
For each pixel in the image, the LIMOs collect photons in four 2 ns-wide consecutive
